PROJECT SUMMARY/ABSTRACT Vesicle transport within the Golgi apparatus is a useful system for studying the targeting specificity of intracellular transport vesicles. The mammalian Golgi consists of four to eight cisternae, each of these containing a unique set of protein- and lipid-modifying enzymes. Most of the transport through the Golgi occurs by cisternal maturation, which suggests that secretory proteins always remain within the cisternae, while resident enzymes are recycled via retrograde vesicles. This sorting model implies that several distinct types of retrograde vesicles (with differing enzyme content) generate the Golgi?s differential enzyme distribution. The central coordinator for retrograde vesicle tethering at the Golgi is the evolutionarily conserved oligomeric Golgi (COG) complex. The COG complex consists of eight gene products, each of which is critical for the Golgi functions. The COG complex functions in the tethering of vesicles that recycle Golgi glycosylation enzymes residing in cis, medial, and trans compartments. Consequently, defects in seven COG subunits have been identified in patients with congenital disorders of glycosylation type II. The COG complex is required for a broad range of essential processes, including protein and lipid glycosylation, sorting and retrograde vesicular trafficking, but the exact mechanism of COG function is an enigma. Detailed analyses of HEK293T and HeLa knock-out (KO) cell lines depleted of individual COG subunits have revealed that each subunit is indispensable to the stability and function of the entire COG complex. Further, a complete COG complex is essential for the stability of a subset of Golgi enzymes, but nonessential for another subset, indicating the existence of both COG-dependent and COG-independent Golgi recycling pathways. We propose that the COG complex orchestrates local recycling of a subset of Golgi enzymes via multipronged interaction with specific SNAREs, Rabs, and coiled-coil tethering factors. COG malfunction results in a rapid loss and degradation of COG- dependent vesicles, triggering upregulation of selective compensatory mechanisms (modulation of cholesterol biosynthesis, Golgi perinuclear repositioning, and modification of the endocytic pathway) which are essential for survival of COG-deficient human cells. To test this hypothesis, first we will utilize gene-edited cell lines and a combination of biochemical and microscopy tools to investigate the molecular details of the COG complex? dependent enzyme-recycling pathway and the COG complex?independent pathway (Aim 1). Next, we will reconstitute COG complex vesicle-tethering activity in vitro (Aim 2). Finally, we will investigate novel stress and compensatory mechanisms that allow for the survival of COG-deprived human cells (Aim 3). The existence of several independent Golgi recycling mechanisms is likely to be important for the elasticity of eukaryotic secretory and endocytic pathways. Understanding how the COG complex spatially and temporally controls the precise tethering of selected transport vesicles is critical to our understanding of membrane trafficking and protein glycosylation in human cells.